Difference between revisions of "Team:Lambert GA/Demonstrate"

To demonstrate the in-field application of this water filtration method, Lambert iGEM replicated the process using water from a nearby creek. The water was disturbed, spiked with an e.coli liquid culture, and run through filtration bottles. The filtered water was then plated on amp plates and cultures were grown in order to see how effective the bottles were at filtering e.coli. Due to cholera being larger than e.coli, the material that was able to filter out the majority of e.coli cells should be able to filter out cholera.

Biosensor Cells

Freeze Drying Protocol

1. Add 5 ml of sterile LB media and 5ul of chloramphenicol and ampicillin to a culture tube.
2. Obtain the desired plate. Take 1-3 colonies from the biosensor cells’ plate with a sterilized inoculating loop. Stir vigorously to ensure colonies are dispensed into solution.
3. Place culture tubes on a rack in the incubator. Set to shake and temperature to 36-38 C, and grow overnight.
4. Centrifuge liquid culture and discard the supernatant. Resuspend the pellet in 5ml of Microbial Freeze Drying Buffer with tryptic soy broth.
5. Aliquot 500ul of the suspension into sterile vials with stopper.
6. Turn on the lyophilizer and start the condenser. Set the shelf to 4°C.
7. Center the vials on the shelf. Either manually or with programmed controls, freeze the samples down to -40°C. This should take about 30-60 minutes, but it is very dependent upon the lyophilizer. If the rate of freezing can be controlled, a practical rate is to drop the temperature by 1°C per minute. The samples should be visually frozen.
8. Let the samples sit at -40°C for 1 hour to complete freezing.
9. Turn on the vacuum pump. Within 10-20 minutes, the vacuum should be under 200 millitorr (mtorr).
10. After the vacuum is below 200 mtorr, increase the temperature of the shelf for primary drying. The temperature can be up to -15°C. Let continue overnight.
11. For second drying, raise the shelf temperature to 20°C and dry for 2 hours.
12. With the stoppering mechanism, pit the stoppers on the vacuum. Turn off vacuum.
13. Store at 4°C in the dark.

In-Field Preparation of Cells
*Tubes should be on ice at all times unless centrifuging*

1. Resuspend in 5 ml of LB agar. Incubate for two hour.
2. Aliquot 1 ml of that liquid culture into 4 ml of sterile LB.
3. Shake in an incubator overnight. If no incubator present, let sit at room temperature for a day and a half.
4. Add the 5 ml liquid culture into 500 ml of sterile LB.
5. Using the portable spectrophotometer, check the optical density every hour. Continue growing until the OD is 0.6.
6. Pour cells into 50 ml conical tubes, on ice. Keep there for 30 minutes.
7. Pipet up the bottom 1.5 ml in the tube and transfer to a microcentrifuge tube.
8. With the 3D-fuge, centrifuge cells for 20 minutes, discard supernatant.
9. Add 1.5 ml of ice-cold sterile into the microcentrifuge to resuspend. Add into empty 50 ml conical tube.
10. Add another 11.5 ml of ice-cold sterile milliQ water to each conical tube, pipet up and down.
11. Combine samples into 2 total conical tubes.
12. Let sit on ice for 30 minutes. Take bottom 1.5 ml of each sample and transfer into a empty microcentrifuge tube.
13. With 3D-fuge, centrifuge for 20 minutes, discard supernatant, and repeat steps 9 and 10.
14. Let sit for 30 minutes on ice.
15. Transfer bottom 1.5 ml into a microcentrifuge tube. Centrifuge for 20 minutes, discard supernatant, and add 1.5 ml of ice-cold 10% glycerol.
16. Transfer the microcentrifuge into an empty conical tube. Add 23.5 ml of ice-cold 10% glycerol.
17. Let sit for 30 minutes on ice. Repeat steps 15 and 16.
18. Let sit for 30 minutes on ice. Take bottom 1.5 ml and transfer into a microcentrifuge tube. Centrifuge with 3D-fuge for 20 minutes.
19. Resuspend in 1.5 ml of ice-cold 10% glycerol. Transfer into conical tube and add 2.5 ml of ice-cold 10% glycerol.
20. Aliquot 1 ml into microcentrifuge tubes. Store in -80 freezer.

In recognition of the dire crisis cholera outbreaks have presented to the world, inflicting harm on millions and killing hundreds of thousands, we present a new solution to ensure a proactive response to cholera outbreaks can be mounted by targeting contamination within the water sources. CAPTIVATE ™ constitutes a portable diagnostic kit to test the water in local areas to determine whether Vibrio Cholerae is present or not. This diagnostic kit includes Lambert iGEM’s hardware components such as the 3-D Fuge, Chrome-Q, Chrome-Q Base, and the Electropen System. The kit also contains the wet-lab components including the Spira Swab (gauze water filter), freeze-dried Biosensor cells in culture tubes, and a RNA extraction kit. The kit is capable of powering a phone for the Color-Q app and a cooling system needed for certain wet-lab parts of the project through a solar panel. CAPTIVATE ™ portable diagnostic kit could work in conjunction of existing World Health Organization treatment kits which can provide a more efficient assessment to determine if cholera exists in that water source.

Materials

• Scoop (collection jar-disposable water bottle)
• Muslin (Muslin will be placed in the 100 mL Nalgene bottle filled with diH2O: This diH2O can be obtained from boiling the water when sterilizing the muslin cloths).
• 100 ml Nalgene bottle with 2cm.hole at the bottom (will be wrapped around with aluminum foil to keep sterile)
• Sterile 100 mL Nalgene bottle
• Crochet Hook, or other straight stick that can be sterilized and reused
• lysozyme in TE buffer
• proteinase K
• Rneasy Mini Kit
• 1M HCl
• 10 ml sterile distilled H2O (1ml needed for spin columns, and disposal of biosensor cells)
• Electropen System (Cuvette and Electropen)
• X-gal
• Freeze-Dried Cells in a vial
• LB in vials
• Culture Tubes
• Microcentrifuge Tubes
• PCR Tubes
• 50 ml conical tubes
• Lego spectrophotometer
• 1% sodium hydrochlorite
• 10% glycerol
• Chrome-Q & Chrome-Q base
• Color-Q app/phone connected to the solar panel.
• 3-d fuge
• pipette/micropipette